Immunofluorescence Tests

Biobest Laboratories produces immunofluorescence assay (IFA) test plates and slides by immobilising cells infected with virus or parasitic tachyzoites by chemical fixation. Samples are added to the test plates or slides at several dilutions and incubated at core body temperature; if antibodies to the antigen are present in the sample they will bind to the antigen during this incubation (Figure 1). The tests are washed to remove any antibodies which have not bound then a suitable anti-species antibody conjugated to a fluorescent marker is added. this secondary antibody will bind to any antibodies which were present in the sample and had bound to the antigen. The tests are incubated then washed again before being read by microscopy using ultra violet illumination. The fluorescent marker glows with a bright apple green fluorescence under the UV light indicating the presence of specific antibodies bound to the antigen (figure 2). The pattern of fluorescence observed often confirms the specificity of the reaction. The sample antibody titre is the last dilution of the sample to show this specific fluorescence.

Figure 1. Detection of Antibodies by immunofluorescence

Test serum is incubated with antigen immobilised on a 96-well plate or microscope slide

Secondary antibodies labelled with a fluorescence are added

After washing, any bound secondary antibodies can be detected by shining UV light on the slide. The label used may be a fluorochrome (IFA test) or be an enzyme which induces a colour change when the substrate is added (ELISA).

Figure 2. FCoV Serology by IFA

An FCoV infected culture of cells is used to show the presence of antibodies to the virus in cat serum. Infected cells fluoresce against a background of uninfected cells. In cats without antibodies to FCoV a uniform background is seen.